人前列腺素E1检测方法
时间：2012-04-25 阅读：3392
产品特例：人前列腺素E1（PGE1）ELISA Kit
英文名：human Prostaglandin E1,PG-E1 ELISA Kit
PGE1 EIA kit
Catalog No. ADI-900-005
96 Well Kit
Table of Contents
Description Page 2
Introduction 2
Precautions 2
Materials Supplied 3
Storage 3
Materials Needed but Not Supplied 3
Sample Handling 4
Procedural Notes 5
Reagent Preparation 5
Assay Procedure 6
Calculation of Results 7
Typical Results 7
Typical Standard Curve 8
Typical Quality Control Parameters 8
Performance Characteristics 9
Sample Dilution Recommendations 11
References 11
Limited Warranty 12
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Description
The PGE1 EIA kit is a competitive immunoassay for the quantitative determination of Prostaglandin E1 in biological fluids. Please read the complete kit insert before performing this assay. The EIA kit uses a polyclonal antibody to PGE1 to bind, in a competitive manner, the PGE1 in the sample or an alkaline phosphatase molecule which has PGE1 covalently attached to it. After a simultaneous incubation at room temperature the excess reagents are washed away and substrate is added. After a short incubation time the enzyme reaction is stopped and the yellow color generated is read on a microplate reader at 405nm. The intensity of the bound yellow color is inversely proportional to the concentration of PGE1 in either standards or samples. The measured optical density is used to calculate the concentration of PGE1. For further explanation of the principles and practice of immunoassays please see the excellent books by Chard1 or Tijssen2.
Introduction
Prostaglandin E1 （PGE1 ） is synthesized from DGLA, dihomo-γ-linolenic acid3. PGE1 has been shown to have a number of biological actions, including vasodilation4, proliferation of vascular smooth muscle cells5, plaet aggregation6 and has been shown to have insulin-like actions7,8. Its effects are induced by receptor mediated elevation of cAMP9. It is the major prostaglandin in semen10,11.
Prostaglandin E1
H
OH
Precautions
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
1 Some kit components contain azide, which may react with lead or copper plumbing. When disposing of reagents always flush with large volumes of water to prevent azide build-up.
2 Stop Solution is a solution of trisodium phosphate. This solution is caustic; care should be taken in use.
3 The activity of the alkaline phosphatase conjugate is dependent on the presence of Mg2+ and Zn2+ ions. The activity of the conjugate is affected by concentrations of chelators （>10 mM） such as EDTA and EGTA.
4 We test this kit’s performance with a variety of samples, however it is possible that high levels of interfering substances may cause variation in assay results.
5 The Prostaglandin E1 Standard provided, Catalog No. 80-0085, is supplied in ethanolic buffer at a pH optimized to maintain PGE1 integrity. Care should be taken handling this material because of the known and unknown effects of prostaglandins.
Materials Supplied
1. Donkey anti-Sheep IgG Microtiter Plate, One Plate of 96 Wells, Catalog No. 80-0045
A plate using break apart strips coated with donkey antibody specific to sheep IgG.
2. PGE1 EIA Conjugate, 6 mL, Catalog No. 80-0094
A blue solution of alkaline phosphatase conjugated with PGE1.
3. PGE1 EIA Antibody, 6 mL, Catalog No. 80-0095
A yellow solution of a sheep polyclonal antibody to PGE1 .
4. Assay Buffer, 30 mL, Catalog No. 80-0010
Tris buffered saline, containing proteins and sodium azide as preservative.
5. Wash Buffer Concentrate, 30 mL, Catalog No. 80-1286
Tris buffered saline containing detergents.
6. Prostaglandin E1 Standard, 0.5 mL, Catalog No. 80-0085
A solution of 50,000 pg/mL PGE1.
7. pNpp Substrate, 20 mL, Catalog No. 80-0075
A solution of p-nitrophenyl phosphate in buffer. Ready to use.
8. Stop Solution, 6 mL, Catalog No. 80-0247 A solution of trisodium phosphate in water. Keep tightly capped. Caution: Caustic.
9. PGE1 Assay Layout Sheet, 1 each, Catalog No. 30-0016
10. Plate Sealer, 1 each, Catalog No. 30-0012
Storage All components of this kit, except the PGE1 Conjugate, are stable at 4 °C until the kit’s expiration date. The PGE1 Conjugate must be stored at -20 °C.
Materials Needed but Not Supplied
1 Deionized or distilled water.
2 Precision pipets for volumes between 5 µL and 1,000 µL.
3 Repeater pipets for dispensing 50 µL and 200 µL.
4 Disposable beakers for diluting buffer concentrates.
5 Graduated cylinders.
6 A microplate shaker.
7 Adsorbent paper for blotting.
8 Microplate reader capable of reading at 405 nm, preferably with correction between 570 and 590 nm.
Sample Handling
The PGE1 EIAkit is compatible with PGE1 samples in a wide range of matrices after dilution in Assay Buffer. Please refer to the Sample Recovery recommendations on page 11 for details of suggested dilutions. However, the end user must verify that the recommended dilutions are appropriate for their samples. Samples containing sheep IgG may interfere with the assay.
Samples in the majority of tissue culture media, including those containing fetal bovine serum, can also be read in the assay, provided the standards have been diluted into the tissue culture media instead of Assay Buffer. There will be a small change in binding associated with running the standards and samples in media. Users should only use standard curves generated in media or buffer to calculate concentrations of PGE1 in the appropriate matrix. For tissue, urine and plasma samples, prostaglandin synthetase inhibitors, such as, indomethacin or meclofenamic acid at concentrations up to 10 µg/mL should be added to either the tissue homogenate or urine and plasma samples.
Some samples normally have very low levels of PGE1 present and extraction may be necessary for accurate measurement. A suitable extraction procedure is outlined below:
Materials Needed
1 PGE1 Standard to allow extraction efficiency to be accuray determined.
2 2M hydrochloric acid, deionized water, ethanol, hexane and ethyl acetate.
3 200 mg C18 Reverse Phase Extraction Columns.
Procedure
1 Acidify the plasma, urine or tissue homogenate by addition of 2M HCl to pH of 3.5. Approximay 50 µL of HCl will be needed per mL of plasma. Allow to sit at 4 °C for 15 minutes. Centrifuge samples in a microcentrifuge for 2 minutes to remove any precipitate.
2 Prepare the C18 reverse phase column by washing with 10 mL of ethanol followed by 10 mL of deionized water.
3 Apply the sample under a slight positive pressure to obtain a flow rate of about 0.5 mL minute. Wash the column with 10 mL of water, followed by 10 mL of 15% ethanol, and finally 10 mL hexane. Elute the sample from the column by addition of 10 mL ethyl acetate.
4 If analysis is to be carried out immediay, evaporate samples under a stream of nitrogen. Add at least 250 µL of Assay Buffer to the dried samples. Vortex well then allow to sit for five minutes. Repeat twice more. If analysis is to be delayed, store samples as the eluted ethyl acetate solutions at -80 °C until the immunoassay is to be run. Evaporate the organic solvent under a stream of nitrogen prior to running assay and reconstitute as above
Please refer to references 12-15 for details of extraction protocols.
Procedural Notes
1 Do not mix components from different kit lots or use reagents beyond the kit expiration date.
2 Allow all reagents to warm to room temperature for at least 30 minutes before opening.
3 Standards can be made up in either glass or plastic tubes.
4 Pre-rinse the pipet tip with reagent, use fresh pipet tips for each sample, standard and reagent.
5 Pipet standards and samples to the bottom of the wells.
6 Add the reagents to the side of the well to avoid contamination.
7 This kit uses break-apart microtiter strips, which allow the user to measure as many samples as desired. Unused wells must be kept desiccated at 4 °C in the sealed bag provided. The wells should be used in the frame provided.
8 Care must be taken to minimize contamination by endogenous alkaline phosphatase. Contaminating alkaline phosphatase activity, especially in the substrate solution, may lead to high blanks. Care should be taken not to touch pipet tips and other items that are used in the assay with bare hands.
9 Prior to addition of substrate, ensure that there is no residual wash buffer in the wells. Any remaining wash buffer may cause variation in assay results.
Reagent Preparation
1. PGE1 Standard Allow the 50,000 pg/mL PGE1 standard solution to warm to room temperature. Label six 12 x 75 mm glass tubes #1 through #6. Pipet 1mL of standard diluent （Assay Buffer or Tissue Culture Media） into tube #1. Pipet 750 µL of standard diluent into tubes #2 through #6. Remove 100 µL of diluent from tube #1. Add 100 µL of the 50,000 pg/mL standard to tube #1. Vortex thoroughly. Add 250 µL of tube #1 to tube #2 and vortex thoroughly. Add 250 µL of tube #2 to tube #3 and vortex. Continue this for tubes #4 through #6.
The concentration of PGE1 in tubes #1 through #6 will be 5,000, 1,250, 313, 78.1, 19.5 and 4.88 pg/mL respectively. See PGE1 Assay Layout Sheet for dilution details.
Diluted standards should be used within 60 minutes of preparation.
2. Wash Buffer Just before use, prepare the Wash Buffer by diluting 5 mL of the supplied concentrate with 95 mL of deionized water. Discard unused buffer or add up to 0.09% sodium azide （w/v） for storage.
Assay Procedure Bring all reagents to room temperature for at least 30 minutes prior to opening.
All standards and samples should be run in duplicate.
1. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells with the desiccant back into the pouch and seal the ziploc. Store unused wells at 4 °C.
2. Pipet 100 µL of standard diluent （Assay Buffer or Tissue Culture Media） into the NSB and the Bo （0 pg/mL Standard） wells.
3. Pipet 100 µL of Standards #1 through #6 into the appropriate wells.
4. Pipet 100 µL of the Samples into the appropriate wells.
5. Pipet 50 µL of Assay Buffer into the NSB wells.
6. Pipet 50 µL of blue Conjugate into each well, except the TA and Blank wells.
7. Pipet 50 µL of yellow Antibody into each well, except the Blank, TA and NSB wells.
NOTE: Every well used should be Green in color except the NSB wells which should be Blue. The Blank and TA wells are empty at this point and have no color.
8. Incubate the plate at room temperature on a plate shaker for 2 hours at ~500 rpm. The plate may be covered with the plate sealer provided, if so desired.
9. Empty the contents of the wells and wash by adding 400 µL of wash solution to every well. Repeat the wash 2 more times for a total of 3 Washes.
10. After the final wash, empty or aspirate the wells, and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer.
11. Add 5 µL of the blue Conjugate to the TA wells.
12. Add 200 µL of the pNpp Substrate solution to every well. Incubate at room temperature for 45 minutes without shaking.
13. Add 50 µL of Stop Solution to every well. This stops the reaction and the plate should be read immediay.
14. Blank the plate reader against the Blank wells, read the optical density at 405 nm, preferably with correction between 570 and 590 nm. If the plate reader is not able to be blanked against the Blank wells, manually subtract the mean optical density of the Blank wells from all readings.
Calculation of Results
Several options are available for the calculation of the concentration of PGE1 in the samples. We recommend that the data be handled by an immunoassay software package utilizing a 4 parameter logistic curve fitting program. If data reduction software is not readily available, the concentration of PGE1 can be calculated as follows:
1. Calculate the average net Optical Density （OD） bound for each standard and sample by subtracting the average NSB OD from the average OD bound: Average Net OD = Average Bound OD -Average NSB OD
2. Calculate the binding of each pair of standard wells as a percentage of the maximum binding wells （Bo）, using the following formula: Percent Bound = Net OD x 100 Net Bo OD
3. Using Logit-Log paper plot Percent Bound versus Concentration of PGE1 for the standards. Approximate a straight line through the points. The concentration of PGE1 in the unknowns can be determined by interpolation.
Typical Results The results shown below are for illustration only and should not be used to calculate results from another assay. Mean Average Percent PGE1
Sample OD （-Blank） Net OD Bound （pg/mL）
Blank OD （0.072）
TA 0.848 0.848
NSB 0.000 0.000 0.00%
Bo 0.621 0.621 100% 0
S1 0.100 0.100 16.0% 5,000
S2 0.184 0.184 29.5% 1,250
S3 0.291 0.291 46.6% 312.5
S4 0.436 0.436 69.9% 78.1
S5 0.557 0.557 89.3% 19.5
S6 0.597 0.597 95.7% 4.88
Unknown 1 0.379 0.379 60.7% 143
Unknown 2 0.199 0.199 31.9% 894 Typical Standard Curve A typical standard curve is shown below. This curve must not be used to calculate PGE1 concentrations; each user must run a standard curve for each assay.
Typical Quality Control Parameters
Total Activity Added = 0.848 x 10 = 8.48 %NSB = 0.0% %Bo/TA = 7.4% Quality of Fit = 0.999 （Calculated from 4 parameter logistic curve fit）
20% Intercept = 2,988 pg/mL 50% Intercept = 268 pg/mL 80% Intercept = 39 pg/mL
Performance Characteristics
The following parameters for this kit were determined using the guidelines listed in the National Committee for Clinical Laboratory Standards （NCCLS） Evaluation Protocols16.
Sensitivity
Sensitivity was calculated by determining the average optical density bound for twenty （20） wells run as Bo, and comparing to the average optical density for twenty （20） wells run with Standard #6. The detection limit was determined as the concentration of PGE1 measured at two （2） standard deviations from the zero along the standard curve.
Average Optical Density for the Bo = 0.579 ± 0.012 （2.07%） Average Optical Density for Standard #6 = 0.558 ± 0.009 （1.61%）
Delta Optical Density （0-4.88 pg/mL） = 0.021 2 SD’s of the Zero Standard = 2 x 0.012 = 0.024
Sensitivity = 0.024 x 4.88 pg/mL = 5.58 pg/mL
0.021
Linearity
A sample containing 50,000 pg/mL PGE1 was diluted 7 times 1:2 in the kit Assay Buffer and measured in the assay. The data was plotted graphically as actual PGE1 concentration versus measured PGE1 concentration.
The line obtained had a slope of 1.0681 and a correlation coefficient of 1.000.
Precision
Intra-assay precision was determined by taking samples containing low, medium and high concentrations of PGE1 and running these samples multiple times （n=24） in the same assay. Inter-assay precision was determined by measuring three samples with low, medium and high concentrations of PGE1 in multiple assays （n=8）.
The precision numbers listed below represent the percent coefficient of variation for the concentrations of PGE determined in these assays as calculated by a 4 parameter logistic curve fitting program.
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Cross Reactivities
The cross reactivities for a number of related eicosanoid compounds was determined by dissolving the cross reactant （purity checked by N.M.R. and other analytical methods） in Assay Buffer at concentrations from 500,000 to 4.8 pg/mL. These samples were then measured in the PGE1 assay, and the measured PGE1 concentration at 50% B/Bo calculated. The % cross reactivity was calculated by comparison with the actual concentration of cross reactant in the sample and expressed as a percentage.
Compound Cross Reactivity PGE1 100% PGE2 6.50% PGE3 2.22% 13,14-dihydro-PGE1* 1.50% PGE0 1.45% 15-keto-PGE1* 1.15% 13,14-dihydro-15-keto-PGE1* 0.19% PGF1α 0.14% PGF2α 0.04% 6-keto-PGF1α <0.1% PGA2 <0.1% PGD2 <0.1% PGB1 <0.1% 13,14-dihydro-15-keto-PGF2α <0.1% 6,15-keto-13,14-dihydro-PGF1α <0.1% Thromboxane B2 <0.1% Misoprostol <0.1% 2-Arachidonoylglycerol <0.1% Anandamide <0.1%
* Data from Covance Laboratories, Inc., Vienna, Virginia.
Sample Recoveries
Please refer to pages 4 and 5 for Sample handling recommendations and Standard preparation.
PGE1 concentrations were measured in a variety of different samples including tissue culture media, human saliva, serum, plasma, and urine. For samples in tissue culture media, ensure that the standards have been diluted into the same media （refer to page 4）. PGE1 was spiked into the undiluted samples of these media which were then diluted with the kit Assay Buffer and then assayed in the kit. The following results were obtained:
Sample % Recovery Recommended Dilution*
Tissue Culture Media 90-110 None Human Saliva 107.2 1:10 Human Urine 109.9 1:50 Human Serum 87.0 1:20 Human Plasma 107.7 1:20
* See Sample Handling instructions on page 4 for details.
References
1. T. Chard,”An Introduction to Radioimmunoassay & Related Techniques 4th Ed.”, （1990） Amsterdam: Elsevier.
2. P. Tijssen,”Practice & Theory of Enzyme Immunoassays”, （1985） Amsterdam: Elsevier.
3. J. Mai, et al., Prostaglandins., （1980） 20: 187.
4. A.G. Olsson and L.A. Carlson,”Advances in Prostaglandin and Thromboxane Research.”, （1976） NY: Raven Press.
5. D.G. Cornwell, et al., Lipids, （1979） 14: 194-207.
6. A.J. Ally and D.F. Horrobin, Prostaglandins and Medicine., （1980） 4: 431-438.
7. H.A. Haessler and J.D. Crawford, J. Clin. Invest., （1967） 46: 1065-69.
8. D.F. Horrobin and M.S. Manku, International Prostaglandin Conference, （1979） 53.
9. N.P. Kurstjens, et al., Biochem. Biophys. Res. Comm., （1990） 167: 1162.
10. L. Speroff, et al., Am. J. Obstet. Gynec.., （1978） 107: 1111.
11. D. Rubin & M. Laposata., J. Biol. Chem., （1991） 226: 23618.
12. K. Green, et al., Anal. Biochem, （1973） 54: 434.
13. J. Frolich, et al., J. Clin. Invest., （1975） 55: 763.
14. J.E. Shaw & P.W. Ramwell, Meth. Biochem. Anal., （1969） 17: 325.
15. K. Green, et al., Adv. Prostaglandin & Thromboxane Res., （1978） 5: 15.
16. National Committee for Clinical Laboratory Standards Evaluation Protocols, SC1,（1989） Villanova, PA: NCCLS.
Use of Product
This product contains research chemicals. As such, they should be used and handled only by or under the supervision of technically qualified indi-viduals. This product is not intended for diagnostic or human use.
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Warranty
Enzo Life Sciences International, Inc. makes no warranty of any kind, expressed or implied, which extends beyond the description of the product in this brochure, except that the material will meet our specifications at the time of delivery. Enzo Life Sciences International, Inc. makes no guarantee of results and assumes no liability for injuries, damages or penalties resulting from product use, since the conditions of handling and use are beyond our control.
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